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µ-Crystallin is a NADPH-regulated thyroid hormone binding protein encoded by the CRYM gene in humans. It is primarily expressed in the brain, muscle, prostate, and kidney, where it binds thyroid hormones, which regulate metabolism and thermogenesis. It also acts as a ketimine reductase in the lysine degradation pathway when it is not bound to thyroid hormone. Mutations in CRYM can result in non-syndromic deafness, while its aberrant expression, predominantly in the brain but also in other tissues, has been associated with psychiatric, neuromuscular, and inflammatory diseases. CRYM expression is highly variable in human skeletal muscle, with 15% of individuals expressing ≥13 fold more CRYM mRNA than the median level. Ablation of the Crym gene in murine models results in the hypertrophy of fast twitch muscle fibers and an increase in fat mass of mice fed a high fat diet. Overexpression of Crym in mice causes a shift in energy utilization away from glycolysis towards an increase in the catabolism of fat via β-oxidation, with commensurate changes of metabolically involved transcripts and proteins. The history, attributes, functions, and diseases associated with CRYM, an important modulator of metabolism, are reviewed.

μ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T3 and T4. Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym. We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6–147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, β-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that μ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of μ-crystallin are associated with a shift toward greater fat metabolism., Graphical abstract Image, Highlights • μ-Crystallin (Crym) is expressed specifically in transgenic skeletal muscle at highly elevated levels. • T3 is increased ~190 fold in the Tibialis anterior muscle of Crym tg mice. • Small but significant changes in gene and protein expression in tg muscle towards a slow twitch, oxidative phenotype. • Metabolic studies show that Crym tg mice increase their use of fat as an energy source. • Female Crym tg mice gain weight faster on high fat or simple carbohydrate diets than controls.

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3′‐triiodo‐l‐thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ‐Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRβ) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage‐specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3‐ and androgen‐mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth., What's new? Thyroid hormones may play a role in the progression of prostate cancer (PCa). In this study, the authors found that PCa cells had decreased expression of the thyroid‐hormone‐binding protein μ‐Crystallin (CRYM). Lower CRYM was also associated with poor prognosis in men with PCa. In addition, CRYM inhibited thyroid‐hormone and androgen signaling, as well as 18F‐fluoromethylcholine uptake by PCa cells. These results suggest that CRYM may act as a novel antagonist against factors that fuel PCa progression, and therefore thyroid signalling may offer a new therapeutic target in slowing aggressive prostate cancer.

Previous studies have identified ~50 genes that contribute to non-syndromic autosomal dominant sensorineural deafness (DFNA). However, in numerous families with hearing loss, the specific gene mutation remains to be identified. In the present study, the clinical characteristics and gene mutations were analyzed in a Chinese pedigree with hereditary hearing loss. The clinical characteristics of the family members were assessed and a detailed audiology function examination was performed. Whole-exome sequencing (WES) was performed to identify the gene mutation responsible for the hearing loss. Sanger sequencing was used to verify the candidate mutation detected in the family. The family consisted of 31 members, seven of whom were diagnosed with sensorineural deafness of varying degrees. No mutation was identified by the general deafness gene chip. However, a novel heterozygous mutation in exon 3 (c.152C>T; Pro51Leu) of the gene crystallin µ (CRYM) was identified by WES. This result was further verified by Sanger sequencing. Co-segregation of genotypes and phenotypes suggested that this novel mutation was instrumental for the hearing loss/DFNA. In conclusion, the present study identified a novel pathogenic mutation, NM_001888.5(CRYM): c.152C>T(Pro51Leu), associated with DFNA. This mutation has not been reported previously and further functional studies are warranted.

The formation process of the Crimean Tatar theater can be divided into the following periods: medieval – folk theater (the initial round dance and toy puppet theater of shadows “Karagoz”, the theater of one actor “meddah”, the arena theater “orta oyuny”); Khan`s theater in the middle of the XVIII century (penetration into the Crimea of European theater traditions in the era of the Crimean Khan Crym Girey I); the revival of traditions of the Crimean Tatar theater late XIX–XX centuries (the activities of a theater-goers group of the Jadidist Crimean Tatar youth–followers of I. Gasprinsky, under the leadership of J. Meinov – the efforts of the Crimean Tatar noblewoman-myrzachkas under the leadership of A. Taiganskaya; organization of a professional Simferopol Tatar theater troupe under the People’s Commissar of Education of the Crimean ASSR in 1921 and creation and activities of the Crimean Tatar Drama Theater, headed by A. Taigan, and the Crimean Tatar amateur movement in the Crimea, and among the Crimean Tatar foreign diaspora of 1923–1944 (Soviet pre-deportation period); recreation and current activities of the Crimean Tatar theater in the Crimea,1989 (post deportation period). In this article, for the first time in the art history, is revealed the so-called Khan`s period in the formation of the Crimean Tatar theater, discussed the revolutionary activity in the field of Crimean Tatar art, the ascetic activity of the Crimean Khan Crym Girey I to promote the ideas of European theater traditions and create a classical theater in the Crimea. The picture of the actions undertaken by the Crimean ruler in the construction of theater business in the Crimea, as well as his thoughts and statements about the theater, was recreated according to the text published in the XVIII century, memories of personal meetings and conversations with Crym Girey I of European authors: German – von der Goltz, Polish – Pilshtynova, Russian – Nikiforov, Frenchman – de Tott, Austrian – Kleeman. Based on these recollections is built a clear and explicit picture of a role of Crym Girey I as a pioneer in bringing European theater traditions and creation of a classical theater in the culture of the Crimea, the Turkic and Muslim worlds.

The proteins sodium iodide symporter (NIS), μ-crystallin (CRYM), and thyroid hormone receptor beta (THRB) have been associated with prognosis in various cancer entities. While NIS and THRB may serve as possible therapeutic targets, the role of CRYM in cancer is still unclear. Protein levels of 44 patients with adenoid cystic carcinoma of the head and neck were analyzed using immunohistochemistry and correlated with clinicopathological data and outcome. NIS was positive in 72%, CRYM was positive in 55%, and THRB was positive in 39% of the patients. CRYM-positive adenoid cystic carcinomas were associated with a better cause-specific survival. Thus, our data indicate that CRYM might be a suitable positive prognostic marker in adenoid cystic carcinoma of the head and neck. Furthermore, expression of NIS was present in most patients and therefore evaluation of the use of radioiodine treatment is recommended.

Objective: To study the role of long non-coding RNA (lncRNA) CRYM-AS1 in human gastric cancer. Methods: Expression levels of CRYM-AS1 in cell lines and clinical tissues were examined by RT-qPCR. The association between CRYM-AS1 levels and clinicopathological parameters / survival rates of gastric cancer patients was analyzed.Cell functional experiments including MTT assay, glucose consumption / lactate production / ATP production detection were performed to examine the role of CRYM-AS1 in cell aerobic glycolysis and cell proliferation of gastric cancer cells. Subcellular fractionation location detection, western blot, RIP (RNA binding protein immunoprecipitation) assay, CHIP (Chromatin immunoprecipitation) assay and BSP (Bisulfite sequencing PCR) assay were carried out to explore the molecular mechanism of CRYM-AS1 in gastric cancer cells.Results: CRYM-AS1 was low expressed in gastric cancer cells and tissues compared with normal gastric cells and tissues respectively. CRYM-AS1 was negatively correlated with TNM staging, tumor size and overall survival (OS) rate in gastric cancer patients. CRYM-AS1 inhibited gastric cancer cell aerobic glycolysis and cell proliferation. CRYM-AS1 directly bound to EZH2 and mediated the CRYM promoter methylation and consequently negatively regulated the expression of CRYM. Forced expression of CRYM rescued the decreased aerobic glycolysis and cell proliferation induced by CRYM-AS1 in gastric cancer cells.Conclusion: CRYM-AS1 was an important biomarker and could be used for human gastric cancer treatment.

Background Tumor‐educated platelets (TEPs) may enable blood‐based cancer diagnosis. This study aimed to identify diagnostic TEPs genes involved in carcinogenesis. Materials and Methods The TEPs differentially expressed genes (DEGs) between healthy samples and early/advanced cancer samples were obtained using bioinformatics. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were used to identify the pathways and functional annotation of TEPs DEGs. Protein‐protein interaction of these TEPs DEGs was analyzed based on the STRING database and visualized by Cytoscape software. The correlation analysis and diagnostic analysis were performed to evaluate the diagnostic value of TEPs mRNAs expression for early/advanced cancers. Quantitative real‐time PCR (qRT‐PCR) was applied to validate the role of DEGs in cancers. Results TEPs mRNAs were mostly involved in protein binding, extracellular matrix, and cellular protein metabolic process. RSL24D1 was negatively correlated to early‐stage cancers compared to healthy controls and may be potentially used for early cancer diagnosis. In addition, HPSE, IFI27, LGALS3BP, CRYM, HBD, COL6A3, LAMB2, and IFITM3 showed an upward trend in the expression from early to advanced cancer stages. Moreover, ARL2, FCGR2A, and KLHDC8B were positively associated with advanced, metastatic cancers compared to healthy controls. Among the 12 selected DEGs, the expression of 7 DEGs, including RSL24D1, IFI27, CRYM, HBD, IFITM3, FCGR2A, and KLHDC8B, were verified by the qRT‐PCR method. Conclusion This study suggests that the 7‐gene TEPs liquid‐biopsy biomarkers may be used for cancer diagnosis and monitoring., Receiving operating characteristic (ROC) curves show the relative diagnostic performance of TEPs RSL24D1, HPSE, IFI27, LGALS3BP, CRYM, HBD, COL6A3, LAMB2, IFITM3, ARL2, FCGR2A, and KLHDC8B have predictive validation value for cancers.

The molecular mechanism underlying embryonic implantation is vital to understand the correct communications between endometrium and developing conceptus during early stages of pregnancy. This study’s objective was to determine molecular changes in the uterine endometrial proteome during the preimplantation and peri-implantation between 9 days (9D), 12 days (12D), and 16 days (16D) of pregnant Polish Large White (PLW) gilts. 2DE-MALDI-TOF/TOF and ClueGOTM approaches were employed to analyse the biological networks and molecular changes in porcine endometrial proteome during maternal recognition of pregnancy. A total of sixteen differentially expressed proteins (DEPs) were identified using 2-DE gels and MALDI-TOF/TOF mass spectrometry. Comparison between 9D and 12D of pregnancy identified APOA1, CAPZB, LDHB, CCT5, ANXA4, CFB, TTR upregulated DEPs, and ANXA5, SMS downregulated DEPs. Comparison between 9D and 16D of pregnancy identified HP, APOA1, ACTB, CCT5, ANXA4, CFB upregulated DEPs and ANXA5, SMS, LDHB, ACTR3, HP, ENO3, OAT downregulated DEPs. However, a comparison between 12D and 16D of pregnancy identified HP, ACTB upregulated DEPs, and CRYM, ANXA4, ANXA5, CAPZB, LDHB, ACTR3, CCT5, ENO3, OAT, TTR down-regulated DEPs. Outcomes of this study revealed key proteins and their interactions with metabolic pathways involved in the recognition and establishment of early pregnancy in PLW gilts.

The potential benefits of treating subclinical hypothyroidism (SCH) are unclear and still controversial. Thus, we surgically induced SCH in rats and evaluated the effects of thyroxine (T4) replacement on the gene expression levels of deiodinases and thyroid hormone (TH) transporters in different tissues. SCH was induced by hemithyroid electrocauterization. The control animals underwent the same surgical procedure but were not subjected to electrocauterization (sham). After 14 days, half of the SCH animals were treated with T4 (SCH + T4). At the end of the experimental protocol, all of the rats were euthanized, serum hormone concentrations were measured, and RNA analyses were performed on different tissues and organs. Consistent with previous studies, we observed increased TSH levels, normal TH levels, and reduced hypothalamic TRH expression in the SCH group. Additionally, Dio2 mRNA expression was downregulated in the hippocampus and pituitary, and Dio1 was upregulated in the kidney and pituitary of the SCH animals. The changes in Dio3 expression were tissue-specific. Concerning TH transporters, Mct10 expression was upregulated in the pituitary, kidney, hypothalamus, and hippocampus, and Mct8 expression was downregulated in the kidney of the SCH group. Crym expression was upregulated in the kidney and pituitary. Notably, T4 replacement significantly attenuated serum TSH levels and reverted Dio1, Dio2, Mct10, and Crym expression in the pituitary, hippocampus, and kidney to levels that were similar to the sham group. Tissue-specific responses were also observed in the liver and hypothalamus. Our results indicate that treatment of SCH should be considered before the appearance of clinical symptoms of hypothyroidism.

Adolescence is a sensitive window for reward- and stress-associated behavior. Although stress during this period causes long-term changes in behavior in males, how females respond is relatively unknown. Here we show that social isolation stress in adolescence, but not adulthood, induces persistent but opposite effects on anxiety- and cocaine-related behaviors in male vs. female mice, and that these effects are reflected in transcriptional profiles within the adult medial amygdala (meA). By integrating differential gene expression with co-expression network analyses, we identified crystallin mu (Crym), a thyroid-binding protein, as a key driver of these transcriptional profiles. Manipulation of Crym specifically within adult meA neurons recapitulates the behavioral and transcriptional effects of social isolation and re-opens a window of plasticity that is otherwise closed. Our results establish that meA is essential for sex-specific responses to stressful and rewarding stimuli through transcriptional programming that occurs during adolescence.

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3’-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidence indicates a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRa/TRb) in target tissues. We show in this study that low CRYM expression levels in PCa patient samples are associated with early BCR and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in PET/MRI imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3 and androgen-mediated signalling. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth.HighlightsThyroid and androgen hormone driven pathways in prostate cancer (PCa) are antagonized by μ- Crystallin (CRYM).[18F]fluoromethylcholine uptake and prognostic values in PCa correlate with CRYM protein levels.Reduced CRYM expression predicts early biochemical recurrence (BCR) in PCa patients.

Chronic Hepatitis C virus (HCV) infection still constitutes a major global health problem with almost half a million deaths per year. To date, the human hepatoma cell line Huh7 and its derivatives is the only cell line that robustly replicates HCV. However, even different subclones and passages of this single cell line exhibit tremendous differences in HCV replication efficiency. By comparative gene expression profiling using a multi-pronged correlation analysis across eight different Huh7 variants, we identified 34 candidate host factors possibly affecting HCV permissiveness. For seven of the candidates, we could show by knock-down studies their implication in HCV replication. Notably, for at least four of them, we furthermore found that overexpression boosted HCV replication in lowly permissive Huh7 cells, most prominently for the histone-binding transcriptional repressor THAP7 and the nuclear receptor NR0B2. For NR0B2, our results suggest a finely balanced expression optimum reached in highly permissive Huh7 cells, with even higher levels leading to a nearly complete breakdown of HCV replication, likely due to a dysregulation of bile acid and cholesterol metabolism. Our unbiased expression-profiling approach, hence, led to the identification of four host cellular genes that contribute to HCV permissiveness in Huh7 cells. These findings add to an improved understanding of the molecular underpinnings of the strict host cell tropism of HCV.

The protein μ-crystallin (CRYM) is a novel component of the marsupial lens that has two functions: it is a key regulator of thyroid hormone transportation and a reductase of sulfur-containing cyclic ketimines. In this study, we examined changes of the expression pattern of CRYM in different rat organs during development using immunohistochemistry and immunoblotting. As CRYM is reportedly expressed in the corticospinal tract, we also investigated CRYM expression in human cases of amyotrophic lateral sclerosis (ALS) using immunohistochemistry. In the rat brain, CRYM was expressed in the cerebral cortex, basal ganglia, hippocampus and corticospinal tract in the early postnatal period. As postnatal development progressed, CRYM expression was restricted to large pyramidal neurons in layers V and VI of the cerebral cortex and pyramidal cells in the deep layer of CA1 in the hippocampus. Even within the same regions, CRYM-positive and negative neurons were distributed in a mosaic pattern. In the kidney, CRYM was expressed in epithelial cells of the proximal tubule and mesenchymal cells of the medulla in the early postnatal period; however, CRYM expression in the medulla was lost as mesenchymal cell numbers decreased with the rapid growth of the medulla. In human ALS brains, we observed marked loss of CRYM in the corticospinal tract, especially distally. Our results suggest that CRYM may play roles in development of cortical and hippocampal pyramidal cells in the early postnatal period, and in the later period, performs cell-specific functions in selected neuronal populations. In the kidney, CRYM may play roles in maturation of renal function. The expression patterns of CRYM may reflect significance of its interactions with T3 or ketimines in these cells and organs. The results also indicate that CRYM may be used as a marker of axonal degeneration in the corticospinal tract.

Gene mutation has an important role in disease pathogenesis; therefore, genetic screening is a useful tool for diagnosis. The present study screened pathogenic genes, ectodysplasin A (EDA) and lamin A/C (LMNA), in a patient with suspected syndromic hearing impairment and various other symptoms including tooth and skin abnormalities. Large‑scale sequencing of 438 deafness‑associated genes and whole‑genome sequencing was also performed. The present findings did not identify copy number variation and mutations in EDA; therefore, excluding the possibility of EDA‑initiated ectodermal dysplasia syndrome. A synonymous mutation in LMNA, possibly due to a splicing abnormality, did not elucidate the pathogenesis of Hutchinson‑Gilford progeria syndrome. Whole‑genome sequencing revealed copy number variations or mutations in various candidate genes which may elucidate part of the symptoms observed. The copy number variations and mutations were also used to identify single nucleotide variations (SNVs) in crystallin mu (CRYM), RAB3 GTPase activating protein catalytic subunit 1 (RAB3GAP1) and Wnt family member 10A (WNT10A), implicated in deafness, hypogonadism and tooth/skin abnormalities, respectively. The importance of an existing SNV in CRYM and a novel SNV in RAB3GAP1 in pathogenesis remains to be further elucidated. The WNT10A p.G213S mutation was confirmed to be the etiological cause of tooth agenesis and ectodermal dysplasia as previously described. It was concluded that a mutation in WNT10A may be the reason for some of the symptoms observed in the patient; however, other genes may also be involved for other symptoms. The findings of the present study provide putative gene mutations that require further investigation in order to determine their roles in pathogenesis.

© 2019 Lassaletta, Calvino, Morales-Puebla, Lapunzina, Rodriguez-de la Rosa, Varela-Nieto and Martinez-Glez., Vestibular schwannomas (VSs) are benign tumors composed of differentiated neoplastic Schwann cells. They can be classified into two groups: sporadic VS and those associated with neurofibromatosis type 2 (NF2). VSs usually grow slowly, initially causing unilateral sensorineural hearing loss (HL) and tinnitus. These tumors cause HL both due to compression of the auditory nerve or the labyrinthine artery and due to the secretion of different substances potentially toxic to the inner ear or the cochlear nerve. As more and more patients are diagnosed and need to be managed, we are more than ever in need of searching for biomarkers associated with these tumors. Owing to an unknown toxic substance generated by the tumor, HL in VS may be linked to a high protein amount of perilymph. Previous studies have identified perilymph proteins correlated with tumor-associated HL, including μ-Crystallin (CRYM), low density lipoprotein receptor-related protein 2 (LRP2), immunoglobulin (Ig) γ-4 chain C region, Ig κ-chain C region, complement C3, and immunoglobulin heavy constant γ 3. Besides, the presence of specific subtypes of heat shock protein 70 has been suggested to be associated with preservation of residual hearing. It has been recently demonstrated that chemokine receptor-4 (CXCR4) is overexpressed in sporadic VS as well as in NF2 tumors and that hearing disability and CXCR4 expression may be correlated. Further, the genetic profile of VS and its relationship with poor hearing has also been studied, including DNA methylation, deregulated genes, growth factors, and NF2 gene mutations. The knowledge of biomarkers associated with VS would be of significant value to maximize outcomes of hearing preservation in these patients., This work was supported by a FEDER/Ministerio de Economía y Competitividad grant (SAF2017-86107-R) and by a Comunidad Autónoma de Madrid grant (B2017/BMD-3688).

The claustrum is a subcortical nucleus that exhibits dense connectivity across the neocortex. Considerable recent progress has been made in establishing its genetic and anatomical characteristics, however a core, contentious issue that regularly presents in the literature pertains to the rostral extent of its anatomical boundary. The present study addressed this issue in the rat brain.Using a combination of immunohistochemistry and neuronal tract tracing, we have examined the expression profiles of several genes that have previously been identified as exhibiting a differential expression profile in the claustrum relative to the surrounding cortex. The expression profiles of parvalbumin, crystallin mu (Crym), and guanine nucleotide binding protein (G protein), gamma 2 (Gng2) were assessed immunohistochemically alongside, or in combination with cortical anterograde, or retrograde tracer injections. Retrograde neuronal tracer injections into various thalamic nuclei were used to further establish the rostral border of the claustrum.Expression of all three markers delineated a nuclear boundary that extended considerably (~500 μm) beyond the anterior horn of the neostriatum. Cortical retrograde and anterograde neuronal tracer injections, respectively, revealed distributions of cortically-projecting claustral neurons and cortical efferent inputs to the claustrum that overlapped with the gene marker-derived claustrum boundary. Finally, retrograde tracer injections centred in nucleus reuniens, whilst including the rhomboid, mediodorsal and centromedial nuclei, revealed that insular cortico-thalamic projections encapsulated a claustral area, with strongly diminished cell label, that corresponded to the claustrum.

Objective: Cochlear implantation is the most important treatment currently available for profound sensorineural hearing loss. The aim of this study was to investigate the etiology of hearing loss in patients with cochlear implantation, and to compare outcomes. Methods: Japanese hearing loss patients who received cochlear implants (CIs) or electric acoustic stimulation (EAS) in Shinshu University hospital (n = 173, prelingual onset: 92, postlingual onset: 81) participated in this study. Invader assay followed by the targeted exon-sequencing of 63 deafness genes using Massively parallel DNA sequencing (MPS) was applied. For prelingual patients, additional imaging examination, cCMV screening, and pediatric examination were performed for precise diagnosis. Results: Genetic screening successfully identified the causative mutation in 60% of patients with prelingual onset hearing loss and in 36% of those with postlingual hearing loss. Differences in the kinds of genes identified were observed between the two groups. Although there were marked variations in the outcome of cochlear implantation, patients with specific deafness gene mutations showed relatively good results. Conclusion: The present study showed genetic etiology is a major cause of hearing loss in CI/EAS patients. Patients possessing mutations in a number of deafness genes known to be expressed within inner ear have achieved satisfactory auditory performance, suggesting that the identification of the genetic background facilitates the prediction of post-CI performance. MPS is a powerful tool for the identification of causative deafness genes in patients receiving cochlear implantation. Therefore, determination of the involved region inside/outside of the cochlea by identification of the responsible gene is essential.

Transport of thyroid hormone (TH) across the plasma membrane is necessary for the genomic action of T3 mediated by its nuclear T3 receptor. MCT8 and MCT10 have been identified as important TH transporters. Mutations in MCT8 result in severe psychomotor retardation. In addition to TH transport into the cell, MCT8 and MCT10 also facilitate TH efflux from cells. Therefore, the aim of this study was to examine if MCT8 and MCT10 increase the availability of T3 for its nuclear receptor rather than generate a rapid equilibrium between cellular and serum T3. T3 action was investigated in JEG3 cells co-transfected with TRβ1 and a T3 response element-driven luciferase construct, and T3 metabolism was analyzed in cells transfected with type 3 deiodinase (D3). In addition, cells were transfected with MCT8 or MCT10 and/or the cytoplasmic T3-binding protein mu-crystallin (CRYM). Luciferase signal was markedly stimulated by incubating cells for 24 h with 1 nM T3, but this response was not augmented by MCT8 or MCT10 expression. Limiting the time of T3 exposure to 1-6 h and co-transfection with CRYM allowed for a modest increase in luciferase response to T3. In contrast, T3 metabolism by D3 was potently stimulated by MCT8 or MCT10 expression, but it was not affected by expression of CRYM. These results suggest that MCT8 and MCT10 by virtue of their bidirectional T3 transport have less effect on steady-state nuclear T3 levels than on T3 levels at the cell periphery where D3 is located. CRYM alters the dynamics of cellular TH transport but its exact function in the cellular distribution of TH remains to be determined.

The thyroid hormone-binding protein μ-crystallin (CRYM) mediates thyroid hormone action by sequestering triiodothyronine in the cytoplasm and regulating the intracellular concentration of thyroid hormone. As thyroid hormone action is closely associated with glycolipid metabolism, it has been proposed that CRYM may contribute to this process by reserving or releasing triiodothyronine in the cytoplasm. We aimed to clarify the relationship between CRYM and glycolipid metabolism by comparing wild-type and CRYM knockout mice fed a high-fat diet. Each group was provided a high-fat diet for 10 weeks, and then their body weight and fasting blood glucose levels were measured. Although no difference in body weight was observed between the two groups with normal diet, the treatment with a high-fat diet was found to induce obesity in the knockout mice. The knockout group displayed increased dietary intake, white adipose tissue, fat cell hypertrophy, and hyperglycemia in the intraperitoneal glucose tolerance test. In CRYM knockout mice, liver fat deposits were more pronounced than in the control group. Enhanced levels of PPARγ, which is known to cause fatty liver, and ACC1, which is a target gene for thyroid hormone and is involved in the fat synthesis, were also detected in the livers of CRYM knockout mice. These observations suggest that CRYM deficiency leads to obesity and lipogenesis, possibly in part through increasing the food intake of mice fed a high-fat diet.

Mammalian ketimine reductase is identical to μ-crystallin (CRYM)-a protein that is also an important thyroid hormone binding protein. This dual functionality implies a role for thyroid hormones in ketimine reductase regulation and also a reciprocal role for enzyme catalysis in thyroid hormone bioavailability. In this research we demonstrate potent sub-nanomolar inhibition of enzyme catalysis at neutral pH by the thyroid hormones L-thyroxine and 3,5,3'-triiodothyronine, whereas other thyroid hormone analogues were shown to be far weaker inhibitors. We also investigated (a) enzyme inhibition by the substrate analogues pyrrole-2-carboxylate, 4,5-dibromopyrrole-2-carboxylate and picolinate, and (b) enzyme catalysis at neutral pH of the cyclic ketimines S-(2-aminoethyl)-L-cysteine ketimine (owing to the complex nomenclature trivial names are used for the sulfur-containing cyclic ketimines as per the original authors' descriptions) (AECK), Δ(1)-piperideine-2-carboxylate (P2C), Δ(1)-pyrroline-2-carboxylate (Pyr2C) and Δ(2)-thiazoline-2-carboxylate. Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain. In silico docking of various ligands into the active site of the X-ray structure of the enzyme suggests an unusual catalytic mechanism involving an arginine residue as a proton donor. Given the critical importance of thyroid hormones in brain function this research further expands on our knowledge of the connection between amino acid metabolism and regulation of thyroid hormone levels.

μ-Crystallin (CRYM) is also known as NADPH-dependent cytosolic T3-binding protein. A study using CRYM-null mice suggested that CRYM stores triiodothyronine (T3) in tissues. We previously established CRYM-expressing cells derived from parental GH3 cells. To examine the precise regulation of T3-responsive genes in the presence of CRYM, we evaluated serial alterations of T3-responsive gene expression by changing pericellular T3 concentrations in the media. We estimated the constitutive expression of three T3-responsive genes, growth hormone (GH), deiodinase 1 (Dio1), and deiodinase 2 (Dio2), in two cell lines. Subsequently, we measured the responsiveness of these three genes at 4, 8, 16, and 24 h after adding various concentrations of T3. We also estimated the levels of these mRNAs 24 and 48 h after removing T3. The levels of constitutive expression of GH and Dio1 were low and high in C8 cells, respectively, while Dio2 expression was not significantly different between GH3 and C8 cells. When treated with T3, Dio2 expression was significantly enhanced in C8 cells, while there were no differences in GH or Dio1 expression between GH3 and C8 cell lines. In contrast, removal of T3 retained the mRNA expression of GH and Dio2 in C8 cells. These results suggest that CRYM expression increases and sustains the T3 responsiveness of genes in cells, especially with alteration of the pericellular T3 concentration. The heterogeneity of T3-related gene expression is dependent on cellular CRYM expression in cases of dynamic changes in pericellular T3 concentration.

The lysine catabolism pathway differs in adult mammalian brain from that in extracerebral tissues. The saccharopine pathway is the predominant lysine degradative pathway in extracerebral tissues, whereas the pipecolate pathway predominates in adult brain. The two pathways converge at the level of ∆(1)-piperideine-6-carboxylate (P6C), which is in equilibrium with its open-chain aldehyde form, namely, α-aminoadipate δ-semialdehyde (AAS). A unique feature of the pipecolate pathway is the formation of the cyclic ketimine intermediate ∆(1)-piperideine-2-carboxylate (P2C) and its reduced metabolite L-pipecolate. A cerebral ketimine reductase (KR) has recently been identified that catalyzes the reduction of P2C to L-pipecolate. The discovery that this KR, which is capable of reducing not only P2C but also other cyclic imines, is identical to a previously well-described thyroid hormone-binding protein [μ-crystallin (CRYM)], may hold the key to understanding the biological relevance of the pipecolate pathway and its importance in the brain. The finding that the KR activity of CRYM is strongly inhibited by the thyroid hormone 3,5,3'-triiodothyronine (T3) has far-reaching biomedical and clinical implications. The inter-relationship between tryptophan and lysine catabolic pathways is discussed in the context of shared degradative enzymes and also potential regulation by thyroid hormones. This review traces the discoveries of enzymes involved in lysine metabolism in mammalian brain. However, there still remain unanswered questions as regards the importance of the pipecolate pathway in normal or diseased brain, including the nature of the first step in the pathway and the relationship of the pipecolate pathway to the tryptophan degradation pathway.

Thyroid hormones have long been known to play an essential role in brain growth and development, with cytoplasmic thyroid hormone binding proteins (THBPs) playing a critical role in thyroid hormone bioavailability. A major mammalian THBP is μ-crystallin (CRYM), which was originally characterized by its ability to strongly bind thyroid hormones in an NADPH-dependent fashion. However, in 2011 it was discovered that CRYM is also an enzyme, namely ketimine reductase (KR), which catalyzes the NAD(P)H-dependent reduction of -C=N- (imine) double bonds of a number of cyclic ketimine substrates including sulfur-containing cyclic ketimines. The enzyme activity was also shown to be potently inhibited by thyroid hormones, thus suggesting a novel reciprocal relationship between enzyme catalysis and thyroid hormone bioavailability. KR is involved in a number of amino acid metabolic pathways. However, the best documented biological function of KR is its role as a ∆1-piperideine-2-carboxylate (P2C) reductase in the pipecolate pathway of lysine metabolism. The pipecolate pathway is the main L-lysine degradation pathway in the adult brain, whereas the saccharopine pathway predominates in extracerebral tissues and in infant brain, suggesting that KR has evolved to perform specific and important roles in neural development and function. The potent regulation of KR activity by thyroid hormones adds further weight to this suggestion. KR is also involved in L-ornithine/L-glutamate/L-proline metabolism as well as sulfur-containing amino acid metabolism. This review describes the pipecolate pathway and recent discoveries related to mammalian KR function, which have important implications in normal and pathological brain functions.

Hearing impairment is the most frequent sensory deficit in humans. Deafness genes, which harbor pathogenic mutations that have been identified in families with hereditary hearing loss, are commonly expressed in the auditory end organ or the cochlea and may contribute to normal hearing function, yet some of the mouse models carrying these mutations fail to recapitulate the hearing loss phenotype. In this study, we find that distinct expression patterns of those deafness genes in the cochlea of a non-human primate, the common marmoset (Callithrix jacchus). We examined 20 genes whose expression in the cochlea has already been reported. The deafness genes GJB3, CRYM, GRHL2, DFNA5 and ATP6B1 were expressed in marmoset cochleae in patterns different from those in mouse cochleae. Of note, all those genes are causative for progressive hearing loss in humans, but not in mice. The other tested genes, including the deafness gene COCH, in which mutation recapitulates deafness in mice, were expressed in a similar manner in both species. The result suggests that the discrepancy in the expression between rodents and primates may account for the phenotypic difference. This limitation of the rodent models can be bypassed by using non-human primate models such as the marmoset.

The possible neurological and biophysical effects of magnetic fields on animals is an area of active study. Here, we report that courtship activity of male Drosophila increases in a magnetic field and that this effect is regulated by the blue light-dependent photoreceptor cryptochrome (CRY). Naïve male flies exhibited significantly increased courtship activities when they were exposed to a ≥ 20-Gauss static magnetic field, compared with their behavior in the natural environment (0 Gauss). CRY-deficient flies, cryb and crym, did not show an increased courtship index in a magnetic field. RNAi-mediated knockdown of cry in cry-GAL4-positive neurons disrupted the increased male courtship activity in a magnetic field. Genetically expressing cry under the control of cry-GAL4 in the CRY-deficient flies restored the increase in male courtship index that occurred in a magnetic field. Interestingly, artificially activating cry-GAL4-expressing neurons, which include large ventral lateral neurons and small ventral lateral neurons, via expression of thermosensitive cation channel dTrpA1, also increased the male courtship index. This enhancement was abolished by the addition of the cry-GAL80 transgene. Our results highlight the phenomenon of increased male courtship activity caused by a magnetic field through CRY-dependent magnetic sensation in CRY expression neurons in Drosophila.

J. Neurochem. (2011) 118, 379–387. Abstract Ketimine reductase (E.C. 1.5.1.25) was purified to apparent homogeneity from lamb forebrain by means of a rapid multi-step chromatography protocol. The purified enzyme was identified by MS/MS (mass spectrometry) as μ-crystallin. The identity was confirmed by heterologously expressing human μ-crystallin in Escherichia coli and subsequent chromatographic purification of the protein. The purified human μ-crystallin was confirmed to have ketimine reductase activity with a maximum specific activity similar to that of native ovine ketimine reductase, and was found to catalyse a sequential reaction. The enzyme substrates are putative neuromodulator/transmitters. The thyroid hormone 3,5,3′-l-triiodothyronine (T3) was found to be a strong reversible competitive inhibitor, and may have a novel role in regulating their concentrations. μ-Crystallin is also involved in intracellular T3 storage and transport. This research is the first to demonstrate an enzyme function for μ-crystallin. This newly demonstrated enzymatic activity identifies a new role for thyroid hormones in regulating mammalian amino acid metabolism, and a possible reciprocal role of enzyme activity regulating bioavailability of intracellular T3.

BACKGROUND. Androgen receptor (AR) signaling is implicated in prostate cancer progression. Therefore, identification of AR downstream genes is potentially important for selection of novel markers and therapy targets in prostate cancer. METHODS. Expression of a thyroid hormone T3-binding proteinm-crystallin (CRYM) mRNA and protein in cell lines was evaluated by real-time PCR and Western blot, respectively. CRYM expression in vivo was analyzed in patients’ samples by immunohistochemistry. The effects of androgen and T3 on proliferation of MDA PCa 2b cells were assessed by 3 H-thymidine uptake assay. RESULTS. CRYM expression was detected in AR-positive LNCaP and MDA PCa 2b cells. In MDA PCA 2b cells, CRYM was regulated by androgens. Androgen-induced CRYM expression was diminished by antiandrogens or AR siRNA. Inhibition of transcription by a-amanitin caused a reduction in CRYM mRNA. The lack of CRYM expression was noted in LAPC-4 cells and in AR-negative prostate cancer cell lines PC-3 and DU-145. CRYM protein was increased in cancer tissue and decreased in samples from patients after hormonal therapy. In samples from patients with therapy-refractory cancer CRYM was not detectable. We also found that androgens and T3 have additive effects on stimulation of MDA PCa 2b cells proliferation. CONCLUSION. CRYM is a novel androgen-regulated gene whose expression is elevated in prostate cancer but down-regulated in castration therapy-resistant tumors. Prostate 69: 1109–

μ-Crystallin is an NADPH-dependent cytosolic T3-binding protein. A knockout study in mice showed that μ-crystallin has a physiological function as a reservoir of T3 in the cytoplasm in vivo. Patients with nonsyndromic deafness were reported to have point mutations in the μ-crystallin gene. The expression of μ-crystallin is regulated by multiple factors. The present study was performed to determine whether thyroid function is related to the expression of μ-crystallin mRNA in peripheral mononuclear cells. We examined 23 normal healthy male and female subjects and 15 patients with Graves' disease. μ-Crystallin protein expression was determined immunohistochemically in peripheral mononuclear cells. The expression of μ-crystallin mRNA was assessed by reverse transcription of total RNA from peripheral mononuclear cells followed by quantitative PCR. μ-Crystallin protein was detected in peripheral mononuclear cells. The mRNA expression was negatively correlated with age in normal female subjects. The values in female subjects were significantly higher than those in males. The values were positively correlated with serum TSH concentration. The values of the thyrotoxic patients with Graves' disease were lower than those in healthy subjects. A transient increase in μ-crystallin expression was observed within 14-42 days after the initial treatment with antithyroid medication. Thyroid hormone inversely relates to the expression of μ-crystallin mRNA in euthyroid mononuclear cells. Abrupt suppression of thyroid function leads to overexpression of μ-crystallin mRNA in thyrotoxic mononuclear cells. Thyroid hormone-regulated μ-crystallin expression may control thyroid hormone action via the intracytoplasmic T? capacity., Article, HORMONE AND METABOLIC RESEARCH. 41(7):548-553 (2009)

http://www.elsevier.com/wps/find/journaldescription.cws_home/505783/description#description | http://www.elsevier.com/wps/find/journaldescription.cws_home/505783/description#description, Article, TRENDS IN ENDOCRINOLOGY AND METABOLISM. 18(7): 286-289

The pathogenic events that lead to amyotrophic lateral sclerosis (ALS) have not been elucidated. We previously described familial amyotrophic lateral sclerosis (FALS) caused by a Leu126delTT mutation in the Cu/Zn superoxide dismutase gene (SOD1) and have produced transgenic mice (TgM) carrying the same mutation (SOD1(L126delTT) TgM), which exhibited distinct ALS-like motor symptoms and pathological findings. In this study, we analyzed gene expression in the spinal cord of SOD1(L126delTT) TgM by cDNA microarray. Eleven genes were upregulated and two genes downregulated in pre-symptomatic TgM. In post-symptomatic TgM, 54 genes were upregulated and four genes downregulated. We performed real-time polymerase chain reaction (PCR) analysis of 10 of the 54 upregulated genes in the post-symptomatic TgM. The results of real-time PCR were consistent with those obtained by microarray for micro-crystallin (Crym), heat shock protein 1 (Hspb1/HSP27), serine proteinase inhibitor clade A member 3N (Serpina3n), complement component 1q subcomponent beta polypeptide (C1qb), cathepsin H (Ctsh) and polyadenylate binding protein-interacting protein 1 (Paip1). In immunohistochemical analysis, Hsbp1/HSP27 and Ctsh expression levels were increased in reactive astrocytes at the ventral horn of the spinal cord in post-symptomatic TgM, as were Crym, some of Ctsh and Paip1 in microglial cells. Increased expression of those genes was not observed in the control mice. These four genes may be related to the pathogenesis of FALS, especially with regard to the progression of reactive astrocytes and the inflammatory response of microglial cells.

To identify proteins expressed abnormally in facioscapulohumeral muscular dystrophy (FSHD), we extracted soluble proteins from deltoid muscle biopsies from unaffected control and FSHD patients and analyzed them using two-dimensional electrophoresis, mass spectrometry and immunoblotting. Muscles from patients with FSHD showed large increases over controls in a single soluble, 34 kDa protein (pI=5.08) identified by mass spectrometry and immunoblotting as mu-crystallin (CRYM). Soluble fractions of biopsies of several other myopathies and muscular dystrophies showed no appreciable increases in mu-crystallin. Mu-crystallin has thyroid hormone and NADPH binding activity and so may influence differentiation and oxidative stress responses, reported to be altered in FSHD. It is also linked to retinal and inner ear defects, common in FSHD, suggesting that its up-regulation may play a specific and important role in pathogenesis of FSHD.

We have previously shown that follicular thyroglobulin (Tg) has an unexpected function as an autocrine negative-feedback regulator of thyroid hormone (TH) biosynthesis. Tg significantly suppressed the expression of genes necessary for iodide transport and TH synthesis by counteracting stimulation by TSH. However, whether follicular Tg also regulates intracellular TH transport and its secretion from thyrocytes is not known. In the present study, we examined the potential effect of follicular Tg on TH transport and secretion by quantifying the expression of two TH transporters: monocarboxylate transporter 8 (MCT8) and μ-crystallin (CRYM). Our results showed that follicular Tg at physiologic concentrations enhanced both the mRNA and protein expression levels of MCT8 and CRYM in a time- and dose-dependent manner in rat thyroid FRTL-5 cells. Although both the sodium/iodide symporter (NIS), an essential transporter of iodide from blood into the thyroid, and MCT8, a transporter of synthesized TH from the gland, were co-localized on the basolateral membrane of rat thyrocytes in vivo, Tg decreased NIS expression and increased the expression of MCT8 by counteracting TSH action. Thus, the effect of Tg on TH secretion opposed its previously described negative-feedback suppression of TH synthesis. Our results indicate that Tg mediates a complex intrinsic regulation of gene expression that is necessary to balance two opposing vectorial transport systems: the inflow of newly synthesized TH and the outflow of TH by external secretion.

μ-Crystallin (Crym), a thyroid hormone-binding protein, is abnormally up-regulated in the muscles of patients with facioscapulohumeral muscular dystrophy, a dominantly inherited progressive myopathy. However, the physiologic function of Crym in skeletal muscle remains to be elucidated. In this study, Crym was preferentially expressed in skeletal muscle throughout the body. Crym-knockout mice exhibited a significant hypertrophy of fast-twitch glycolytic type IIb fibers, causing an increase in grip strength and high intensity running ability in Crym-null mice. Genetic inactivation of Crym or blockade of Crym by siRNA-mediated knockdown up-regulated the gene expression of fast-glycolytic contractile fibers in satellite cell-derived myotubes in vitro These alterations in Crym-inactivated muscle were rescued by inhibition of thyroid hormone, even though Crym is a positive regulator of thyroid hormone action in nonmuscle cells. The results demonstrated that Crym is a crucial regulator of muscle plasticity, controlling metabolic and contractile properties of myofibers, and thus the selective inactivation of Crym may be a potential therapeutic target for muscle-wasting diseases, such as muscular dystrophies and age-related sarcopenia.-Seko, D., Ogawa, S., Li, T.-S., Taimura, A., Ono, Y. μ-Crystallin controls muscle function through thyroid hormone action.

© 2015. Severed axon tracts fail to exhibit robust or spontaneous regeneration after spinal cord injury (SCI). Regeneration failure reflects a combination of factors, including the growth state of neuronal cell bodies and the regeneration-inhibitory environment of the central nervous system. However, while spared circuitry can be retrained, target reinnervation depends on longitudinally directed regeneration of transected axons. This study describes a biodegradable implant using poly(lactide-co-glycolide) (PLG) bridges as a carrier scaffold to support regeneration after injury. In order to detect regeneration of descending neuronal tracts into the bridge, and beyond into intact caudal parenchyma, we developed a mouse cervical implantation model and employed Crym:GFP transgenic mice. Characterization of Crym:GFP mice revealed that descending tracts, including the corticospinal tract, were labeled by green fluorescent protein (GFP), while ascending sensory neurons and fibers were not. Robust co-localization between GFP and neurofilament-200 (NF-200) as well as GFP and GAP-43 was observed at both the rostral and caudal bridge/tissue interface. No evidence of similar regeneration was observed in mice that received gelfoam at the lesion site as controls. Minimal co-localization between GFP reporter labeling and macrophage markers was observed. Taken together, these data suggest that axons originating from descending fiber tracts regenerated, entered into the PLG bridge at the rostral margin, continued through the bridge site, and exited to re-enter host tissue at the caudal edge of the intact bridge. Finally, regeneration through implanted bridges was associated with a reduction in ipsilateral forelimb errors on a horizontal ladder task.

Recently, crystalized mouse ketimine reductase/CRYM complexed with NADPH was found to have pyruvate bound in its active site. We demonstrate that the enzyme binds α-keto acids, such as pyruvate, in solution, and catalyzes the formation of N-alkyl-amino acids from alkylamines and α-keto acids (via reduction of imine intermediates), but at concentrations of these compounds not expected to be encountered in vivo. These findings confirm that, mechanistically, ketimine reductase/CRYM acts as a classical imine reductase and may explain the finding of bound pyruvate in the crystallized protein.

Human cytosolic 3,5,3'-triiodo-L-thyronine-binding protein, also called mu-crystallin or CRYM, plays important physiological roles in transporting 3,5,3'-triiodo-L-thyronine (T(3)) into nuclei and regulating thyroid-hormone-related gene expression. The crystal structure of human CRYM's bacterial homolog Pseudomonas putida ornithine cyclodeaminase and Archaeoglobus fulgidus alanine dehydrogenase have been available, but no CRYM structure has been reported. Here, we report the crystal structure of human CRYM bound with NADPH refined to 2.6 A, and there is one dimer in the asymmetric unit. The structure contains two domains: a Rossmann fold-like NADPH-binding domain and a dimerization domain. Different conformations of the loop Arg83-His92 have been observed in two monomers of human CRYM in the same asymmetric unit. The peptide bond of Val89-Pro90 is a trans-configuration in one monomer but a cis-configuration in the other. A detailed comparison of the human mu-crystallin structure with its structurally characterized homologs including the overall comparison and superposition of active sites was conducted. Finally, a putative T(3)-binding site in human CRYM is proposed based on comparison with structural homologs.

Differential tissue sensitivity/responsivity to hormones can explain developmental asynchrony among hormone-dependent events despite equivalent exposure of each tissue to circulating hormone levels. A dramatic vertebrate example is during frog metamorphosis, where transformation of the hind limb, brain, intestine, liver, and tail are completely dependent on thyroid hormone (TH) but occurs asynchronously during development. TH transporters (THTs) and cytosolic TH binding proteins (CTHBPs) have been proposed to affect the timing of tissue transformation based on expression profiles and in vitro studies, but they have not been previously tested in vivo. We used a combination of expression pattern, relative expression level, and in vivo functional analysis to evaluate the potential for THTs (LAT1, OATP1c1, and MCT8) and CTHBPs (PKM2, CRYM, and ALDH1) to control the timing of TH-dependent development. Quantitative PCR analysis revealed complex expression profiles of THTs and CTHBPs with respect to developmental stage, tissue, and TH receptor β (TRβ) expression. For some tissues, the timing of tissue transformation was associated with a peak in the expression of some THTs or CTHBPs. An in vivo overexpression assay by tail muscle injection showed LAT1, PKM2, and CRYM increased TH-dependent tail muscle cell disappearance. Co-overexpression of MCT8 and CRYM had a synergistic effect on cell disappearance. Our data show that each tissue examined has a unique developmental expression profile of THTs and CTHBPs and provide direct in vivo evidence that the ones tested are capable of affecting the timing of developmental responses to TH.

Little is known about the right ventricular (RV) proteome in human heart failure (HF), including possible differences compared to the left ventricular (LV) proteome. We used 2-dimensional differential in-gel electrophoresis (pH: 4-7, 10-150 kDa), followed by liquid chromatography tandem mass spectrometry, to compare the RV and LV proteomes in 12 explanted human hearts. We used Western blotting and multiple-reaction monitoring for protein verification and RNA sequencing for messenger RNA and protein expression correlation. In all 12 hearts, the right ventricles (RVs) demonstrated differential expression of 11 proteins relative to the left ventricles (LVs), including lesser expression of CRYM, TPM1, CLU, TXNL1, and COQ9 and greater expression of TNNI3, SAAI, ERP29, ACTN2, HSPB2, and NDUFS3. Principal-components analysis did not suggest RV-versus-LV proteome partitioning. In the nonischemic RVs (n = 6), 7 proteins were differentially expressed relative to the ischemic RVs (n = 6), including increased expression of CRYM, B7Z964, desmin, ANXA5, and MIME and decreased expression of SERPINA1 and ANT3. Principal-components analysis demonstrated partitioning of the nonischemic and ischemic RV proteomes, and gene ontology analysis identified differences in hemostasis and atherosclerosis-associated networks. There were no proteomic differences between RVs with echocardiographic dysfunction (n = 8) and those with normal function (n = 4). Messenger RNA and protein expression did not correlate consistently, suggesting a major role for RV posttranscriptional protein expression regulation. Differences in contractile, cytoskeletal, metabolic, signaling, and survival pathways exist between the RV and the LV in HF and may be related to the underlying HF etiology and differential posttranscriptional regulation.

This study examined whether the AR-CAG repeat length might affect clinical characteristics (testis volume) seminal parameters (sperm count and its mobility) along with hormonal serum profile [FSH, LH, Testosterone (T) and Inhibin B (InhB)] both in idiopathic male infertility (IM) and in infertility due to a previous condition of cryptorchidism (CryM) or to Y chromosome long arm microdeletions (YM).Observational study without intervention(s).One hundred and ten IM patients [90 idiopathic olizoospermic males (IOM) and 20 idiopathic azoospermic males (IAM)], 19 CryM male and 10 YM patients were included. Sixty-one age-matched healthy men who had fathered within 3 years were involved representing the control group (FM).AR-CAG repeats stretch was significantly longer in IOM (p0.05), CryM (p0.05) and YM (p0.001) than FM. When the AR-CAG repeat tracts were subdivided in three subgroups according to the length of CAG repeats tract assessed in fertile subjects (the one with the middle (n 19-21) belonging to the 25 and 75 % inter-quartile, the ends belonging to the25 % inter-quartile and75 % inter-quartile, respectively), there was a statistically significant difference of distribution of AR-CAG tract length among fertile and different groups of infertile men (p=0.0005; chi-square test). Moreover, the subgroup of AR-CAG repeat stretch with 22-28 triplets was associated with lower levels of InhB both in idiopathic oligozoospermic (Scheffe, Bonferroni and Dunett tests p=0.01) and azoospermic men (Scheffe, Bonferroni and Dunett test p=0.05), while, when FM and men with idiopathic infertility were gathered in a single group, both the subgroup of AR- CAG tract with 15-18 repeats and the one with 22-28 repeats are associated with lower testis volume, reduced sperm count and serum InhB levels.Our study showed that the outliers of AR-CAG repeat length seem to influence the function of AR, affecting testis volume and Sertoli cell function and consequently sperm production in both fertile and idiopathic infertile men.

The kidney and several other thyroid hormone-responsive tissues contain a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with an apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T3 and T4 binding. THBP was purified to homogeneity from human kidney cytosol and used to generate proteolytic peptides. Microsequencing of four peptides revealed identity to amino acid sequences deduced from a human cDNA homolog to a cDNA encoding kangaroo mu-crystallin. This protein is a major structural kangaroo lens protein with no known function in other species. A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA ends using a human brain cDNA library and gene-specific PCR primers, confirming identity to the previously cloned human cDNA. The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with significant homologies (40 to 60%) with two bacterial enzymes: lysine cyclodeaminase and ornithine cyclodeaminase. The TH5.9 cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Purified GST fusion protein, but not GST, bound T3 specifically with high affinity [dissociation constant (Kd) = 0.5 nM] in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized [(125)I]T3. T3 binding and photoaffinity labeling of GST fusion protein were activated by NADPH [activation constant (K[act]) = 10(-8) M], but not by NADH. The expressed protein displays the appropriate binding properties, indicating that TH5.9 cDNA encodes the NADP-regulated THBP characterized in human tissues.

My Ph.D. work takes part of a larger project dedicated to the characterization of proteins newly involved into selective expression of certain mouse brain regions. This selective expression being potentially linked to neuronal degeneration associated with neurodegenerative diseases, the corresponding proteins are therefore potential biomarkers. A structural and physico-chemical study has been performed on about ten proteins including CRYM of mouse (mCRYM), which belongs to the Cytosolic-3,3',5-triiodo-L-thyronine(T3)-Binding Protein family since it regulates the concentration of free thyroid hormone,T3, in the cell. mCRYM also belongs to the µ-crystallin family and to the µ-crystallins/Ornithin Cyclodesaminases superfamily. Proteins displaying sequence homologies to these three families of proteins have generally different functions (enzymatic or structural), different tissue localisation and different physico-chemical properties. This diversity is due to the recruitment of genes of the crystalline superfamily to carry different metabolic functions while preserving the taxon-specific crystallins. I have managed to resolve the crystallographic structure of mCRYM in complex with NADP(H) and the thyroid hormone,T3, to 1.75 Å resolution. mCRYM is a very interesting evolution specimen as it belongs to a different family of proteins. However, no enzymatic function has ever been demonstrated for mCRYM. Its structural and thermodynamical characterization has revealed similitudes and divergences with the enzymatic homologues of CRYM and has allowed us to make hypothesis relative to its molecular evolution. These results raise new questions concerning the physiological role of mammalian CRYM: is CRYM an enzyme or a structural protein? How does the NADPH/NADP+ redox couple regulates the genomic and/or non genomic action of the T3 hormone? Is the T3 hormone the only physiological ligand of CRYM in the brain?, Mon projet de thèse s'inscrit dans un vaste projet de caractérisation de protéines nouvellement identifiées dont l'expression est sélective à certaines régions du cerveau. Cette expression sélective pouvant être liée aux phénomènes de dégénérescence neuronale qui caractérisent les maladies neurodégénératives, ces protéines constituent donc des biomarqueurs potentiels. Une étude structurale et physico-chimique a été effectuée sur une dizaine de protéines, dont la protéine CRYM murine (mCRYM) qui fait parti de la famille des Cytosolic- 3,3',5-triiodo-L-thyronine(T3)-Binding Protein car elle régule la concentration en hormone thyroïdienne T3 libre dans la cellule. mCRYM appartient également à la famille des µ-crystallines et à la superfamille des µ-crystallines/Ornithines Cyclodésaminases. Les protéines présentant des homologies pour ces trois familles sont la plupart différentes par leur fonction (enzymatique ou structurale), leur localisation tissulaire et leurs caractéristiques physico-chimiques. Cette diversité est due au recrutement de gènes de la superfamille des crystallines pour diverses fonctions métaboliques tout en conservant le taxon spécifique des crystallines. Je suis parvenue à résoudre sa structure cristallographique complexée au NADP(H) et à l'hormone thyroïdienne T3 à une résolution de 1,75 Å. La protéine mCRYM est un exemple intéressant d'évolution par son appartenance à différentes familles de protéines et, à ce jour, aucune activité enzymatique n'a été identifiée. Sa caractérisation structurale et thermodynamique a donc permis de mettre en évidence les différences et les similitudes avec ses homologues enzymatiques et d'émettre des hypothèses quant à son évolution moléculaire. Ces résultats soulèvent de nouvelles questions concernant son rôle physiologique : mCRYM est-elle une enzyme ou une protéine structurale ? Comment intervient le couple redox NADPH/NADP+ pour réguler l'action génomique et/ou non génomique de l'hormone T3 ? L'hormone T3 est-il le seul ligand physiologique de CRYM dans le cerveau ?

PURPOSE. micro-Crystallin (CRYM) is a major taxon-specific lens protein. The purpose of this study was to investigate the function of CRYM in eyes of mice with endotoxin-induced uveitis (EIU). METHODS. EIU was induced by an injection of a lipopolysaccharide (LPS) into the footpad of male C57BL/6J, CRYM knockout (CRYM(-/-)), and wild-type (CRYM(+/+)) mice. The expression of CRYM in the iris-ciliary body (ICB) was investigated by Western blot analyses and real-time RT-PCR at 12 hours and 1, 3, and 5 days after the LPS injection. The number of cells that had infiltrated the anterior chamber (AC) of the CRYM(+/+) mice was compared to that in the CRYM(-/-) mice at 1, 3, 5, and 7 days. The expressions of the mRNA of interleukin (IL)-1alpha, IL-6, tumor necrosis factor (TNF)-alpha, and granulocyte macrophage-colony stimulating factor (GM-CSF) in the ICB of the two groups of mice were compared. RESULTS. The mRNA of CRYM was upregulated at 12 hours after LPS injection, and CRYM protein increased at 3 days. The number of inflammatory cells in the AC of the CRYM(-/-) mice was not significantly different on day 1 from that in the CRYM(+/+) mice, but was significantly lower (17.9 +/- 1.6 vs. 27.1 +/- 2.4 cells/section) on day 5. Expression of the mRNA of IL-1alpha and -6 in the CRYM(-/-) mice was significantly lower than that in the CRYM(+/+) mice on day 5. CONCLUSIONS. CRYM plays an important role in the development of the second peak of murine EIU.

Transport of (sulfated) iodothyronines across the plasma membrane is required for their intracellular metabolism. Rat Na+/taurocholate cotransporting polypeptide (Ntcp: Slc10al) has been identified as an important transporter protein. We demonstrate that among the 7 members of the solute carrier family SLC10, only human SLC10A1 mediates sodium-dependent transport of the iodothyronine T4 and iodothyronine sulfates T3S and T4S. In contrast to SLC10A2-7, cells co-expressing SLC10A1 and the deiodinase D1 demonstrate a dramatic increase in T3S and T4S metabolism. The SLC10A1 substrates taurocholate, DHEAS and E3S inhibit T3S and T4S transport. Furthermore, co-transfection of SLC10A1 with CRYM,a well known intracellular iodothyronine-binding protein, results in an enhanced intracellular accumulation of T3S and T4S, indicating that CRYM binds iodothyronine sulfates. The present findings indicate that the liver-specific transporter SLC10A1 transports (sulfated) iodothyronines, thereby increasing their intracellular availability. Therefore, SLC10A1 may fulfill a critical step in providing liver D1 with iodothyronine sulfates for rapid degradation. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

During a search for glucose-regulated abundant mRNAs in the diabetic rat kidney, we cloned thyroid hormone binding protein (THBP), also known as μ-crystallin or CRYM. The aim of this study was to investigate the effect of hyperglycemia/high glucose on the expression of THBP. THBP mRNA copy numbers were determined in kidneys and hearts of diabetic GK rats vs normoglycemic Wistar rats, and in human mesangial cells (HMCs) exposed to high glucose using real-time qPCR, and THBP protein levels were measured by Western blotting and immunofluorescence. Intracellular ROS was measured in THBP transfected cells using DCF fluorescence. Hyperglycemia significantly increased THBP mRNA in GK rat kidneys (326 ± 50 vs 147 ± 54, p < 0.05), and hearts (1583 ± 277 vs 191 ± 63, p < 0.05). Moreover, the levels of THBP mRNA increased with age and hyperglycemia in GK rat kidneys, whereas in normoglycemic Wistar rat kidneys there was a decline with age. High glucose significantly increased THBP mRNA (92 ± 37 vs 18 ± 4, p < 0.005), and protein in HMCs. The expression of THBP as a fusion protein in transfected HMCs resulted in reduction of glucose-induced intracellular ROS. We have shown that THBP mRNA is increased in diabetic kidney and heart, is regulated by high glucose in renal cells, and appears to attenuate glucose-induced intracellular ROS. These data suggest that THBP may be involved in the cellular pathways activated in response to glucose. This is the first report linking hyperglycemia with THBP and suggests that the role of THBP in diabetic complications should be further investigated.

usc Serial analysis of gene expression (SAGE), a technique that allows for the simultaneous detection of expression levels of the entire genome without a priori knowledge of gene sequences, was used to examine the transcriptional expression pattern of the Tg2576 mouse model of Alzheimer’s disease (AD). Pairwise comparison between the Tg2576 and nontransgenic SAGE libraries identified a number of differentially expressed genes in the Tg2576 SAGE library, some of which were not previously revealed by the microarray studies. Real-time PCR was used to validate a panel of genes selected from the SAGE analysis in the Tg2576 mouse brain, as well as the hippocampus and temporal cortex of sporadic AD and normal age-matched controls. NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5 (NDUFA5) and FXYD domain-containing ion transport regulator 6 (FXYD6) were found to be significantly decreased in the Tg2576 mouse brain and AD hippocampus. PTEN-induced putative kinase 1 (PINK1), phosphatidylethanolamine binding protein (PEBP), crystalline μ (CRYM), and neurogranin (NRGN) were significantly decreased in AD tissues. The gene ontologies represented in the Tg2576 data were statistically analyzed and demonstrated a significant under-representation of genes involved with G-protein-coupled receptor signaling and odorant binding, while genes significantly over-represented were focused on cellular communication and cellular physiological processes. The novel approach of profiling the Tg2576 mouse brain using SAGE has identified different genes that could subsequently be examined for their potential as peripheral diagnostic and prognostic markers for Alzheimer’s disease. Refereed/Peer-reviewed

The crystallins were discovered more than 100 years ago by Morner (1893. Untersuchungen der Proteinsubstanzen in den lichtbrechenden Medien des Auges. Z. Physiol. Chem. 18, 61-106) as the main structural proteins of the vertebrate eye lens. Since that time the major mammalian crystallins referred to as alpha-, beta-, and gamma-crystallins were characterized with respect to their genetic organization, regulation of their expression pattern and participation in several diseases. In recent years, more and more crystallins have also been identified outside the lens. Evolutionary analysis has demonstrated the relationship of crystallins to proteins involved in protection against stress. The alpha-crystallins form large complexes up to 1Mio Da; they are built up by two subunits referred to as alphaA- and alphaB-crystallins. These subunits are encoded by individual genes, Cryaa and Cryab being localized on different chromosomes and members of the small heat-shock protein family. The alphaA-crystallin is considered to be a molecular chaperone. It is expressed mainly in the lens - mutations in the Cryaa gene lead to recessive or dominant cataracts. In contrast, the alphaB-crystallin is rather ubiquitously expressed; mutations in the Cryab gene are associated with a broad variety of neurological, cardiac and muscular disorders. The beta/gamma-crystallin super family is encoded by at least 14 genes; the proteins are characterized by four Greek key motifs. In mammals, these genes are not only organized as individual genes (Cryba1, Cryba2, Crygf, Crygs, CrygN), but also in duplets (Cryba4-Crybb1 and Crybb2-Crybb3) and in one major cluster (Cryga-Cryge). The various Cryb and Cryg genes are considered to have been evolved by various duplications of the Greek key encoding units. The two main families are distinguished by the fact that each Greek key motif in the Cryb genes is encoded by one exon, whereas two motifs are encoded by one single exon in the Cryg genes. An intermediate between these subfamilies is CrygN encoding the first two Greek key motifs by individual exons, but the others by one single exon. Mutations in the Cryb/Cryg genes lead mainly to an opacification of the eye lens. In some Cryg mutants evidence was presented that the formation of large amyloid-like intranuclear inclusions containing the altered gamma-crystallins is a key event in cataract formation. Cataract formation, caused by Cryg mutations is further characterized by stopping the secondary lens fiber differentiation as indicated by the presence of remnants of cell nuclei, which are usually degraded in secondary fiber cells. Moreover, additional clinical features are being increasingly reported since these crystallins are found outside the eye: the betaB2-crystallin (previously referred to the basic principle crystallin) is also involved in neurogenesis and male infertility. For some of the beta/gamma-crystallins, Ca(2+)-binding properties have been discussed; however, it is an unsolved question whether these crystallins serve as Ca(2+) stores in vivo. Enzyme crystallins are enzymes, which have been recruited to the lens and are expressed there in high concentrations. The mu- and zeta-crystallins (gene symbols: Crym and Cryz, respectively) are discussed as examples for mammals. Mutations in the human CRYM gene lead to non-syndromic deafness, and mutations in the Cryz gene of guinea pigs cause cataracts.

Cellular entry of thyroid hormone is mediated by plasma membrane transporters, among others a T-type (aromatic) amino acid transporter. Monocarboxylate transporter 10 (MCT10) has been reported to transport aromatic amino acids but not iodothyronines. Within the MCT family, MCT10 is most homologous to MCT8, which is a very important iodothyronine transporter but does not transport amino acids. In view of this paradox, we decided to reinvestigate the possible transport of thyroid hormone by human (h) MCT10 in comparison with hMCT8. Transfection of COS1 cells with hMCT10 cDNA resulted in 1) the production of an approximately 55 kDa protein located to the plasma membrane as shown by immunoblotting and confocal microscopy, 2) a strong increase in the affinity labeling of intracellular type I deiodinase by N-bromoacetyl-[125I]T3, 3) a marked stimulation of cellular T4 and, particularly, T3 uptake, 4) a significant inhibition of T3 uptake by phenylalanine, tyrosine, and tryptophan of 12.5%, 22.2%, and 51.4%, respectively, and 5) a marked increase in the intracellular deiodination of T4 and T3 by different deiodinases. Cotransfection studies using the cytosolic thyroid hormone-binding protein μ-crystallin (CRYM) indicated that hMCT10 facilitates both cellular uptake and efflux of T4 and T3. In the absence of CRYM, hMCT10 and hMCT8 increased T3 uptake after 5 min incubation up to 4.0- and 1.9-fold, and in the presence of CRYM up to 6.9- and 5.8-fold, respectively. hMCT10 was less active toward T4 than hMCT8. These findings establish that hMCT10 is at least as active a thyroid hormone transporter as hMCT8, and that both transporters facilitate iodothyronine uptake as well as efflux.

Genes that are highly expressed in the inner ear, as revealed by cDNA microarray analysis, may have a crucial functional role there. Those that are expressed specifically in auditory tissues are likely to be good candidates to screen for genetic alterations in patients with deafness, and several genes have been successfully identified as responsible for hereditary hearing loss. To understand the detailed mechanisms of the hearing loss caused by the mutations in these genes, the present study examined the immunocytochemical localization of the proteins encoded by Crym, KIAA1199 homolog, Uba52, Col9a3, and Col9a1 in the cochlea of rats and mice. Confocal microscopic immunocytochemistry was performed on cryostat sections. Ultrastructurally, postembedding immunogold cytochemistry was applied using Lowicryl sections. Crym protein was predominantly distributed in the fibrocytes in the spiral ligament, as well as the stria vascularis in rats. KIAA1199 protein homolog was localized in various supporting cells, including inner phalangeal, border, inner and outer pillar, and Deiters' cells. Uba52 protein was restrictedly localized within the surface of the marginal cells of the stria vascularis. Collagen type IX was found within the tectorial membrane as well as fibrocytes in the spiral ligament. The present results showed cell-specific localization of the encoded proteins of these highly expressed genes, indicating that the coordinated actions of various molecules distributed in different parts of the cochlea are essential for maintenance of auditory processing in the cochlea.